Project Summary Anxiety and depression are highly prevalent neuropsychiatric disorders which cause substantial morbidity and mortality. A top priority of NIMH is to elucidate the molecules, cells, and neural circuits associated with complex behaviors, including anxiety and depression (NIMH Strategic Objective 1.1). The medial habenula (MHb) is a well-conserved epithalamic structure known to be a powerful modulator of fear and escape behavior in zebrafish and anxiety- and depressive-like behaviors in rodents, and has been shown by MRI to be decreased in volume in humans with depression. The output of MHb neurons is impacted by their synaptic inputs, such as from the medial septum and nucleus of the diagonal band (MSDB), which provides the sole identified GABAergic input to the MHb. We have generated preliminary data showing that CB1 receptor activation by endocannabinoids or by a CB1 receptor agonist suppresses GABAergic inputs in the MHb. Guided by these exciting findings, we hypothesized that 2-AG/CB1 signaling increases the output of MHb neurons via suppression of the MSDB input and disinhibition of MHb neurons, resulting in anxiolytic and antidepressant-like behavioral effects. This hypothesis will be tested via two Specific Aims. In Aim I, we will determine if functional CB1 receptors are expressed on MSDB to MHb axon terminals and how their activation affects MHb neuronal output. We will use viral vectors to express ChR2 in the MSDB and selectively activate MSDB axonal terminals that innervate MHb neurons through optogenetic stimulation. We will then examine whether CB1 receptor activation alters light-induced inhibitory postsynaptic currents (IPSCs) and action potential firing in MHb neurons. In Aim II, we will determine how gain- or loss-of-function in 2-AG/CB1 signaling in the MSDB to MHb circuit affects CB1-mediated suppression of MSDB inputs, and test the hypothesis that this bi-directionally alters anxiety- and depression-like behaviors. Loss-of-function is achieved via targeted deletion of CB1 receptors, while gain-of-function is achieved by targeted deletion of the enzyme that degrades 2-AG, monoacylglycerol lipase. In completing this project, the trainee will master a wide range of powerful experimental techniques, such as ex vivo slice electrophysiology, optogenetics, confocal imaging, in situ hybridization and immunohistochemistry, viral microinjection, pharmacology, and animal behavior assays. Further, the proposed research will expand our knowledge of the neural substrates underlying anxiety and depressive disorders, and how they are regulated by eCB signaling.